Inhibition of T Cell and Promotion of Natural Killer Cell Development by the Dominant Negative Helix Loop Helix Factor Id3

Bipotential T/natural killer (NK) progenitor cells are present in the human thymus. Despite their bipotential capacity, these progenitors develop predominantly to T cells in the thymus. The mechanisms controlling this developmental choice are unknown. Here we present evidence that a member(s) of the family of basic helix loop helix (bHLH) transcription factors determines lineage specification of NK/T cell progenitors. The natural dominant negative HLH factor Id3, which blocks transcriptional activity of a number of known bHLH factors, was expressed in CD34+ progenitor cells by retrovirus-mediated gene transfer. Constitutive expression of Id3 completely blocks development of CD34+ cells into T cells in a fetal thymic organ culture (FTOC). In contrast, development into NK cells in an FTOC is enhanced. Thus, the activity of a bHLH transcription factor is necessary for T lineage differentiation of bipotential precursors, in the absence of which a default pathway leading to NK cell development is chosen. Our results identify a molecular switch for lineage specification in early lymphoid precursors of humans.

Download Full-text

Core binding factors are necessary for natural killer cell development and cooperate with Notch signaling during T-cell specification

Blood ◽

10.1182/blood-2007-10-120261 ◽

2008 ◽

Vol 112 (3) ◽

pp. 480-492 ◽

Cited By ~ 44

Author(s):

Yalin Guo ◽

Ivan Maillard ◽

Sankhamala Chakraborti ◽

Ellen V. Rothenberg ◽

Nancy A. Speck

Keyword(s):

T Cell ◽

Notch Signaling ◽

Natural Killer ◽

Nk Cell ◽

Killer Cell ◽

Cell Development ◽

Lineage Specification ◽

Cell Specification ◽

Developmental Program ◽

Binding Subunit

Abstract CBFβ is the non-DNA binding subunit of the core binding factors (CBFs). Mice with reduced CBFβ levels display profound, early defects in T-cell but not B-cell development. Here we show that CBFβ is also required at very early stages of natural killer (NK)–cell development. We also demonstrate that T-cell development aborts during specification, as the expression of Gata3 and Tcf7, which encode key regulators of T lineage specification, is substantially reduced, as are functional thymic progenitors. Constitutively active Notch or IL-7 signaling cannot restore T-cell expansion or differentiation of CBFβ insufficient cells, nor can overexpression of Runx1 or CBFβ overcome a lack of Notch signaling. Therefore, the ability of the prethymic cell to respond appropriately to Notch is dependent on CBFβ, and both signals converge to activate the T-cell developmental program.

Download Full-text

Functions of E2A-HEB Heterodimers in T-Cell Development Revealed by a Dominant Negative Mutation of HEB

Molecular and Cellular Biology ◽

10.1128/mcb.20.18.6677-6685.2000 ◽

2000 ◽

Vol 20 (18) ◽

pp. 6677-6685 ◽

Cited By ~ 118

Author(s):

Robert J. Barndt ◽

Meifang Dai ◽

Yuan Zhuang

Keyword(s):

T Cell ◽

Target Genes ◽

T Cell Development ◽

Cell Lineage ◽

Cell Receptor ◽

Cell Development ◽

Dominant Negative ◽

Helix Loop Helix ◽

Dominant Negative Mutation ◽

Bhlh Proteins

ABSTRACT Lymphocyte development and differentiation are regulated by the basic helix-loop-helix (bHLH) transcription factors encoded by theE2A and HEB genes. These bHLH proteins bind to E-box enhancers in the form of homodimers or heterodimers and, consequently, activate transcription of the target genes. E2A homodimers are the predominant bHLH proteins present in B-lineage cells and are shown genetically to play critical roles in B-cell development. E2A-HEB heterodimers, the major bHLH dimers found in thymocyte extracts, are thought to play a similar role in T-cell development. However, disruption of either the E2A or HEBgene led to only partial blocks in T-cell development. The exact role of E2A-HEB heterodimers and possibly the E2A and HEB homodimers in T-cell development cannot be distinguished in simple disruption analysis due to a functional compensation from the residual bHLH homodimers. To further define the function of E2A-HEB heterodimers, we generated and analyzed a dominant negative allele of HEB, which produces a physiological amount of HEB proteins capable of forming nonfunctional heterodimers with E2A proteins. Mice carrying this mutation show a stronger and earlier block in T-cell development than HEB complete knockout mice. The developmental block is specific to the α/β T-cell lineage at a stage before the completion of V(D)J recombination at the TCRβ gene locus. This defect is intrinsic to the T-cell lineage and cannot be rescued by expression of a functional T-cell receptor transgene. These results indicate that E2A-HEB heterodimers play obligatory roles both before and after TCRβ gene rearrangement during the α/β lineage T-cell development.

Download Full-text

The Src Family Tyrosine Kinase Fyn Regulates Natural Killer T Cell Development

Journal of Experimental Medicine ◽

10.1084/jem.190.8.1189 ◽

1999 ◽

Vol 190 (8) ◽

pp. 1189-1196 ◽

Cited By ~ 151

Author(s):

Paul Gadue ◽

Neil Morton ◽

Paul L. Stein

Keyword(s):

T Cells ◽

T Cell ◽

Natural Killer ◽

Nk Cell ◽

T Cell Development ◽

Receptor Activation ◽

Cell Development ◽

T Cell Subsets ◽

Nk T Cells ◽

Nk T Cell

T lymphocytes express two Src tyrosine kinases, Lck and Fyn. While thymocyte and T cell subsets are largely normal in fyn−/− mice, animals lacking Lck have impaired T cell development. Here, it is shown that Fyn is required for the rapid burst of interleukin (IL)-4 and IL-13 synthesis, which occurs promptly after T cell receptor activation. The lack of cytokine induction in fyn mutant mice is due to a block in natural killer (NK) T cell development. Studies using bone marrow chimeras indicate that the defect behaves in a cell-autonomous manner, and the lack of NK T cells is probably not caused by inappropriate microenvironmental cues. Both NK T cells and conventional T cells express similar levels of Lck, implying that Fyn and Lck have distinct roles in regulating NK T cell ontogeny. The fyn mutation defines the first signaling molecule that is selectively required for NK T cell, but not for T lymphocyte or NK cell development.

Download Full-text

CD94 1A transcripts characterize lymphoblastic lymphoma/leukemia of immature natural killer cell origin with distinct clinical features

Blood ◽

10.1182/blood-2005-02-0519 ◽

2005 ◽

Vol 106 (10) ◽

pp. 3567-3574 ◽

Cited By ~ 15

Author(s):

Chung-Wu Lin ◽

Ting-Yun Liu ◽

Shee-Uan Chen ◽

Kun-Teng Wang ◽

L. Jeffrey Medeiros ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Killer Cell ◽

Cell Lineage ◽

Lymphoid Cells ◽

Terminal Deoxynucleotidyl Transferase ◽

Gene Rearrangements

AbstractMost lymphoblastic lymphomas (LBLs) are regarded as neoplasms of immature T cells because they express cytoplasmic CD3 and frequently carry T-cell receptor (TCR) gene rearrangements. Immature natural killer (NK) and T cells, however, have a common bipotent T/NK-cell precursor in the thymus, and NK cells also express cytoplasmic CD3. Thus, some LBLs could arise from immature NK cells. Mature NK cells express 2 CD94 transcripts: 1A, induced by interleukin 15 (IL-15), and 1B constitutively. Because immature NK cells require IL-15 for development, CD94 1A transcripts could be a marker of NK-LBL. To test this hypothesis, we used laser capture microdissection to isolate IL-15 receptor α+ lymphoid cells from the thymus and showed that these cells contained CD94 1A transcripts. We then assessed for CD94 transcripts in 21 cases of LBL that were cytoplasmic CD3+, nuclear terminal deoxynucleotidyl transferase positive (TdT+), and CD56-, consistent with either the T-cell or NK-cell lineage. We found that 7 LBLs expressed CD94 1A transcripts without TCR gene rearrangements, suggesting NK-cell lineage. Patients with NK-LBL were younger than patients with T-LBL (15 years versus 33 years; P = .11) and had a better 2-year survival (100% versus 27%; P < .01). These results improve the current classification of LBL and contribute to our understanding of NK-cell differentiation.

Download Full-text

Notch Activation in Cord Blood Progenitors Induces a CD7+ Common NK/T Precursor Capable of NK Cell Commitment without Full Maturation and Differentiation of Lineage Committed Pre-T Lymphocytes with a Th1 Phenotype.

Blood ◽

10.1182/blood.v108.11.644.644 ◽

2006 ◽

Vol 108 (11) ◽

pp. 644-644

Author(s):

Veronika Bachanova ◽

Valarie McCullar ◽

Rosanna Wangen ◽

Jeffrey S. Miller

Keyword(s):

T Cells ◽

T Cell ◽

Cord Blood ◽

Nk Cells ◽

Nk Cell ◽

T Cell Development ◽

Notch Pathway ◽

Cell Development ◽

Dominant Negative ◽

Cell Commitment

Abstract Activation of Notch signaling regulates differentiation and homeostasis of hematopoetic stem cells. After stimulation, intracellular Notch is proteolytically released and by binding the CSL complex and co-activator MAML, and initiates transcription of downstream genes. We hypothesize that Notch is important for distinct stages of lymphoid development. Human cord blood CD34+ progenitor cells were transduced with retrovirus based eGFP-control, eGFP-Notch and Notch Dominant Negative/MAML (eGFP-DN) constructs. CD34+/eGFP+ were sorted and then co-cultured with the mouse embryonic liver cell line EL08.1D2 and exogenous human cytokines (IL-3. IL-7, IL-15, Flt3 ligand and c-kit ligand). As early as 48 hours after transduction, CD34+/Notch+ cells gave rise to population of lymphoid precursors CD34+CD7+CD10- (42±5% of all cells) while essentially no cells with this phenotype were detected with the control or DN construct. Proliferation of eGFP-Notch transduced cells in a 6-day thymidine incorporation assay was higher compared to eGFP-DN transduced cells (8410±839 vs. 1103±209 cpm; n=3; p=0.00005). Within 7 days 11±1.5% NK emerged from CD34+/Notch+cells compared to 0.8±0.2% of CD34+/eGFP+ control cells (n=5, p=0.0001). NK cell generation peaked at day 28 with a significantly higher expression of CD7 on NK cells (Notch: 75±5% vs. eGFP: 4.5±1%, n=5, p=0.00004), and no B lymphocytes were seen. Analysis of Notch induced NK cells demonstrated early expression of L-selectin and increased expression of CD45RA on all lymphoid progenitors. At 4 weeks, functional testing revealed reduced cytotoxicity against K562 (Notch: 37±0.5% vs. eGFP: 63.5±1.3%; n=7, p=0.007) suggesting immature function. CD34+/Notch+ derived NK lymphocytes also showed diminished acquisition of the lectin-type receptor NKG2A (Notch: 8.3±3% vs. eGFP: 27.4±4.5%; p=0.04) and killer immunoglobulin receptors (Notch: 2.2±0.5% vs. eGFP: 10.8±4: p=0.05). We next asked whether the Notch induced CD7+ precursor was NK restricted or a common NK/T cell precursor. After 5 weeks in culture, a distinct population of CD3+ T-cells emerged (Notch: 18±5% vs. eGFP: 1.6±0.2; n=5, p<0.001%) which were CD4 and CD8 negative and did not express surface TCR a/b or g/d, but expressed high levels pre-T-alpha mRNA. These Notch activated cells were bona fide T-cells based on their capacity to produce IL-2 after PMA/Ca/I stimulation (62.4±4% by intracellular staining) while essentially no IL-2 production occurred from eGFP control cells (1.6±0.2%; p<0.0001). T-cell development was dependent on both Notch and the EL08.1D2 as no T-cells resulted from CD34+/eGFP-Notch in the absence of stroma. These findings suggest that in addition to Notch and exogenous cytokines, other soluble factors are required for T cell development. In conclusion, our data showed that activated Notch pathway leads to differentiation of a common CD7+ lymphoid precursor capable of both early NK cell and T-cell differentiation. This suggests that differences in Notch ligands in local microenvironments (marrow, thymus, lymph node) may be an important mechanism to orchestrate NK and T cell development.

Download Full-text

GSK3β Inhibition Promotes T Cell Reconstitution and Preserves Naive T Cell Phenotype in Bone Marrow Reconstituted Mice

Blood ◽

10.1182/blood.v120.21.1890.1890 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1890-1890

Author(s):

Shen Sylvie ◽

Ning Xu ◽

Guy Klamer ◽

Tracey O'Brien ◽

Alla Dolnikov

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

B Cell ◽

Progenitor Cells ◽

Cell Development ◽

Memory T Cell ◽

T Cell Reconstitution ◽

Human T Cells

Abstract Abstract 1890 Stem cell transplantation has become a widely used procedure in the treatment of haematological and non-haematological clinical disorders. Unfortunately, cure is often hampered by relapse of the underlying disease, graft-versus-host disease (GVHD), or severe opportunistic infections. Slow T-cell reconstitution is regarded as primarily responsible for infections, GVHD, and relapse, therefore, enhancing immune reconstitution is important. Glycogen synthase kinase-3β (GSK3β) was recently identified as an important regulator of T cell function acting through the Wingless (Wnt) pathway. The effect of in vivo administration of GSK3β inhibitor 6-Bromoindirubin 3'-oxime (BIO) was examined in a humanised mouse model. Mice transplanted with highly purified cord blood CD34+ stem cells demonstrated efficient multilineage reconstitution including myeloid, B and T cell lymphoid compartments. The presence of human CD4 and CD8 single positive human T cells was abundant in peripheral blood (PB). De novo generated T cells exhibited low CD31 expression in the naïve CD4+ T cells suggesting prolonged post-thymic proliferative history of these cells. This is not completely surprising considering that graft recipient mice are characterized by impaired thymopoiesis following irradiation. Human T cells at various stages of differentiation including late effector T cells were recorded by detecting the expression of CD62L, CD45RA and CD45RO. Late memory T cell skewing was observed in PB and spleen of graft recipient mice. Activation of human T cells expressing CD25 was registered in the spleen, however, the recipients of the graft did not exhibit any signs of GVHD suggesting normal positive and negative selection occurring in the thymus during human T cell development in this mouse model. Human T cells isolated from the spleen of transplanted mice exhibited strong proliferative responses to mitogenic and allogeneic stimulation, however, they did not demonstrated any CTL activity tested following vaccination with human leukaemia U937 cells. In vivo administration of GSK3β inhibitor promoted T cell reconstitution in mice transplanted with human CD34+haematopoietic progenitor cells. Per cell output of T cells from CD34+ and CD34+CD38- primitive bone marrow (BM) progenitor cells was higher in BIO-treated mice while CD19+ B cell output was reduced suggesting T-cell developmental skewing in expense of B cell development. In vitro analysis of CD34+ progenitor cells co-cultured with bone marrow stroma MS5 cells has demonstrated inhibited B-cell development following BIO-treatment. CD31 expression in naïve CD4+ T cells was not up-regulated by BIO suggesting that GSK3β inhibition does not act to increase thymic output of T cells. GSK3β inhibition also increased naïve/memory T cell ratio in reconstituted mice. A similar effect was observed in mice transplanted with mature cord blood (CB)-derived T cells. delayed naive to memory T cell transition is likely related to decreased T cell activation and proliferation demonstrated ex vivo. BIO reduced IFNγ and TNFα production in human T cells. BIO increased naïve T cell production in mitogenically stimulated T cells and in mixed lymphocyte cultures. GSK3β inhibition preserved naïve T cell gene expression profile and suppressed the expression of genes activated during effector T cell differentiation. BIO actrivated β-catenin sigbnaling and up-regulated IL7Rα expression. IL7 signalling prevents activated T cell death following effector differentiation suggesting that the mechanism triggered by BIO may act through the inhibition of activated T cell death. In addition, BIO down-regulated negative regulator of IL7Rα SOCS1 as well as CTLA4 and PDCD1 both up-regulated during effector differentiation. Thus clinically GSK3β inhibition acting to prevent late memory T cell skewing and preserving a subset of naïve T cells may increase T cell diversity and improve T cell responses in the recipients of CB transplant particularly in adult patients with impaired thymic function. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Interleukin-12 inhibits murine graft-versus-host disease

Blood ◽

10.1182/blood.v86.6.2429.bloodjournal8662429 ◽

1995 ◽

Vol 86 (6) ◽

pp. 2429-2438 ◽

Cited By ~ 80

Author(s):

M Sykes ◽

GL Szot ◽

PL Nguyen ◽

DA Pearson

Keyword(s):

T Cells ◽

T Cell ◽

Natural Killer ◽

Graft Versus Host Disease ◽

Killer Cell ◽

Interleukin 12 ◽

Ifn Gamma ◽

Host Disease ◽

Graft Versus Host ◽

Host Type

Interleukin-12 (IL-12) is a potent immunostimulatory cytokine and an inducer of type-1 T-helper cell activity and of cytotoxic T lymphocyte and natural killer cell function. We report here the paradoxical observation that a single injection of 4,900 IU of recombinant murine IL-12 inhibits acute graft-versus-host disease (GVHD) in a fully major histocompatibility complex (MHC) plus multiple minor antigen-mismatched bone marrow transplantation (BMT) model (A/J-->B10). The protective effect was enhanced by administration of T-cell-depleted host-type BM cells, and complete donor-type lymphohematopoietic reconstitution was observed in most animals. Treatment with a protective course of IL-12 led to increased serum interferon-gamma (IFN-gamma) levels as compared with those for GVHD controls at early time points, when IFN-gamma was produced predominantly by host-type natural killer cells, but led to almost complete inhibition of the later GVHD-associated increase in serum IFN-gamma levels, when IFN-gamma is produced predominantly by CD4+ T cells. Furthermore, IL-12 treatment was associated with marked alterations in the kinetics of donor T-cell expansion. Reductions in donor CD4+ and CD8+ T cells were observed in the spleen on day 4 post- BMT, but a marked increase in donor CD8+ cells was observed on day 7. Unlike broadly immunosuppressive methods for inhibiting GVHD, which are associated with loss of antileukemic effects, IL-12 has the potential to mediate antileukemic effects of its own; therefore, the GVHD- inhibitory effects of IL-12 described here suggest a potential application for this cytokine in clinical BMT.

Download Full-text

Adapted NOD/SCID model supports development of phenotypically and functionally mature T cells from human umbilical cord blood CD34+ cells

Blood ◽

10.1182/blood.v99.5.1620 ◽

2002 ◽

Vol 99 (5) ◽

pp. 1620-1626 ◽

Cited By ~ 50

Author(s):

Tessa C. C. Kerre ◽

Greet De Smet ◽

Magda De Smedt ◽

Alfred Zippelius ◽

Mikaël J. Pittet ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Killer Cell ◽

T Cell Development ◽

Cell Development ◽

Scid Mice ◽

Human Umbilical Cord ◽

Low Grade ◽

Hematopoietic Stem ◽

Peripheral T Cells

The NOD-LtSZ scid/scid (NOD/SCID) repopulation assay is the criterion for the study of self-renewal and multilineage differentiation of human hematopoietic stem cells. An important shortcoming of this model is the reported absence of T-cell development. We studied this aspect of the model and investigated how it could be optimized to support T-cell development. Occasionally, low-grade thymic engraftment was observed in NOD/SCID mice or Rag2−/−γc−/− mice. In contrast, the treatment of NOD/SCID mice with a monoclonal antibody against the murine interleukin-2Rβ, (IL-2Rβ) known to decrease natural killer cell activity, resulted in human thymopoiesis in up to 60% of the mice. T-cell development was phenotypically normal and resulted in polyclonal, mature, and functional CD1−TCRαβ+ CD4+ or CD8+single-positive T cells. In mice with ongoing thymopoiesis, peripheral T cells were observed. TREC analysis showed that T cells with a naive phenotype (CD45RA+) emerged from the thymus. In approximately half of these mice, the peripheral T cells included a pauciclonal outgrowth of CD45RO+ cells. These data suggest that all elements of a functional immune system were present in these animals.

Download Full-text

Inflammatory Cytokines Regulate T-Cell Development from Blood Progenitor Cells in a Stage and Dose-Specifc Manner

10.1101/2021.01.18.427186 ◽

2021 ◽

Author(s):

John M. Edgar ◽

Peter W. Zandstra

Keyword(s):

T Cells ◽

T Cell ◽

Progenitor Cells ◽

T Cell Development ◽

Cell Development ◽

Cell Maturation ◽

Hematopoietic Stem ◽

Lineage Differentiation ◽

T Cell Maturation

ABSTRACTT-cell development from hematopoietic stem and progenitor cells (HSPCs) is tightly regulated through Notch pathway activation by the Notch ligands Delta-like (DL) 1 and 4 and Jagged-2. Other molecules, such as stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L) and interleukin (IL)-7, play a supportive role in regulating the survival, differentiation, and proliferation of developing progenitor (pro)T-cells. Numerous other signaling molecules are known to instruct T-lineage development in vivo, but little work has been done to optimize their use for T-cell production in vitro. Using a defined T-lineage differentiation assay consisting of plates coated with the Notch ligand DL4 and adhesion molecule VCAM-1, we performed a cytokine screen that identified IL-3 and tumor necrosis factor α (TNFα) as enhancers of proT-cell differentiation and expansion. Mechanistically, we found that TNFα induced T-lineage differentiation through the positive regulation of T-lineage genes GATA3, TCF7, and BCL11b. TNFα also synergized with IL-3 to induce proliferation by upregulating the expression of the IL-3 receptor on CD34+ HSPCs, yielding 753.2 (532.4-1026.9; 5-95 percentile)-fold expansion of total cells after 14 days compared to 8.9 (4.3-21.5)-fold expansion in conditions without IL-3 and TNFα. We then optimized cytokine concentrations for T-cell maturation. Focusing on T-cell maturation, we used quantitative models to optimize dynamically changing cytokine requirements and used these to construct a three-stage assay for generating CD3+CD4+CD8+ and CD3+CD4−CD8+ T-cells. Our work provides new insight into T-cell development and a robust in vitro assay for generating T-cells to enable clinical therapies for treating cancer and immune disorders.

Download Full-text

Natural killer cell receptors regulate responses of HLA-E–restricted T cells

Science Immunology ◽

10.1126/sciimmunol.abe9057 ◽

2021 ◽

Vol 6 (58) ◽

pp. eabe9057

Author(s):

Lucy C. Sullivan ◽

Thi H.O. Nguyen ◽

Christopher M. Harpur ◽

Sanda Stankovic ◽

Abbie R. Kanagarajah ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Natural Killer Cell ◽

Natural Killer ◽

Killer Cell ◽

Flow Cytometric Analysis ◽

Human Leukocyte ◽

Cell Receptor ◽

Cell Receptors ◽

Natural Killer Cell Receptors

Human cytomegalovirus (CMV) infection can stimulate robust human leukocyte antigen (HLA)–E–restricted CD8+ T cell responses. These T cells recognize a peptide from UL40, which differs by as little as a single methyl group from self-peptides that also bind HLA-E, challenging their capacity to avoid self-reactivity. Unexpectedly, we showed that the UL40/HLA-E T cell receptor (TCR) repertoire included TCRs that had high affinities for HLA-E/self-peptide. However, paradoxically, lower cytokine responses were observed from UL40/HLA-E T cells bearing TCRs with high affinity for HLA-E. RNA sequencing and flow cytometric analysis revealed that these T cells were marked by the expression of inhibitory natural killer cell receptors (NKRs) KIR2DL1 and KIR2DL2/L3. On the other hand, UL40/HLA-E T cells bearing lower-affinity TCRs expressed the activating receptor NKG2C. Activation of T cells bearing higher-affinity TCRs was regulated by the interaction between KIR2D receptors and HLA-C. These findings identify a role for NKR signaling in regulating self/non-self discrimination by HLA-E–restricted T cells, allowing for antiviral responses while avoiding contemporaneous self-reactivity.

Download Full-text